All living organisms emit spontaneous ultraweak photon emission as a result of cellular metabolic processes. In this study, the involvement of reactive oxygen species (ROS) formed as the byproduct of oxidative metabolic processes in spontaneous ultraweak photon emission was studied in human hand skin. The effect of molecular oxygen and ROS scavengers on spontaneous ultraweak photon emission from human skin was monitored using a highly sensitive photomultiplier tube and charged coupled device camera. When spontaneous ultraweak photon emission was measured under anaerobic conditions, the photon emission was decreased, whereas under hyperaerobic condition the enhancement in photon emission was observed. Spontaneous ultraweak photon emission measured after topical application of glutathione, α-tocopherol, ascorbate, and coenzyme Q10 was observed to be decreased. These results reveal that ROS formed during the cellular metabolic processes in the epidermal cells play a significant role in the spontaneous ultraweak photon emission. It is proposed that spontaneous ultraweak photon emission can be used as a noninvasive tool for the temporal and spatial monitoring of the oxidative metabolic processes and intrinsic antioxidant system in human skin.